title: Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.
abstract: Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.
authors.affiliation: Section on Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Reisz
authors.forename: Erika
authors.initials: E
authors.lastname: Bechtold
authors.forename: S Bruce
authors.initials: SB
authors.lastname: King
authors.forename: Leslie B
authors.initials: LB
authors.lastname: Poole
authors.forename: Cristina M
authors.initials: CM
authors.lastname: Furdui
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry / Q000378:metabolism; D003512:Cyclohexanones / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry / Q000378:metabolism; D007460:Iodoacetamide / Q000737:chemistry / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013434:Sulfenic Acids / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003512:Cyclohexanones; D011506:Proteins; D017382:Reactive Oxygen Species; D013434:Sulfenic Acids; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; C004774:dimedone; D003545:Cysteine; D005033:Ethylmaleimide; D001205:Ascorbic Acid; D004229:Dithiothreitol; D007460:Iodoacetamide
keywords: S-nitrosation; dimedone; mass spectrometry; protein sulfenic acid; thiol blocking